The use of antisense oligonucleotides to modulate the expression of genes responsible for malignancy or for resistance to therapy represents an exciting new modality for cancer treatment. However, a major impediment to therapeutic use of antisense oligonucleotides concerns the inefficient transport of these compounds to their sites of action in the cytoplasm and nucleus. It seems likely that the major route of oligonucleotide uptake is via endocytosis. However, endocytotic uptake still puts the oligonucleotide on the "wrong" side of a membrane with respect to its sites of action in the cell, and thus transfer from endosome to cytosol may be a limiting step in antisense pharmacology. The overall goals of this proposal are first, to define and quantitate key aspects of the pathways of cellular uptake and intracellular distribution of antisense oligonucleotides, and second, to design oligonucleotides capable of efficient transport into the cytoplasm and nucleus. (1) Liposomes will be used as a model system to measure permeation rates of oligonucleotides across membranes and the affect of chemical modification on those permeation rates. We will ascertain features of chemically modified oligonucleotides which contribute to rapid passage across membrane barriers. (2) We will define the cellular uptake and subcellular distribution of novel chemically modified oligonucleotides designed for efficient transport. We will use the information generated in Aims 1 and 2 to design modified oligonucleotides that are efficiently taken up by cells, that readily enter the cytoplasm and nucleus, that arc stable, and are thus likely to have a high degree of pharmacological effectiveness. (3) We will test our strategies for enhanced oligonucleotide transport and delivery using P-glycoprotein mediated multi-drug resistance as a demanding experimental model. (4) We will also identify and characterize proteins that are involved in the cell uptake and intracellular distribution of oligonucleotides. The insights gained in these studies should directly contribute to the design of antisense oligonucleotides suitable for effective cancer chemotherapy.